Summary Background Early, sorting endosomes are a major crossroad of membrane traffic, at the intersection of the endocytic and exocytic pathways. The sorting of endosomal cargo for delivery to different subcellular destinations is mediated by a number of distinct coat protein complexes, including AP-1, AP-3 and GGAs. Ultrastructural studies suggest that these coats assemble onto tubular subdomains of the endosomal membrane, but the mechanisms of coat recruitment and assembly at this site remain poorly understood. Results Here we report that the endosomal Rab protein Rab4 orchestrates a GTPase cascade which results in the sequential recruitment of the Arf-like protein Arl1, the Arf-specific guanine nucleotide exchange factors BIG1 and BIG2, and the class I Arfs, Arf1 and Arf3. Knockdown of Arf1, or inhibition of BIG1/2 activity with Brefeldin A results in the loss of AP-1, AP-3 and GGA-3, but not Arl1, from endosomal membranes and the formation of elongated tubules. In contrast, depletion of Arl1 randomizes the distribution of Rab4 on endosomal membranes, inhibits the formation of tubular subdomains and blocks recruitment of BIG1/2, Arfs and adaptor complexes to the endosome. Conclusion Together these findings indicate that Arl1 links Rab4-dependent formation of endosomal sorting domains with downstream assembly of adaptor protein complexes that constitute the endosomal sorting machinery.

The detection of microbes and initiation of an innate immune response occur through pattern recognition receptors (PRRs), which are critical for the production of inflammatory cytokines and activation of the cellular microbicidal machinery. In particular, the production of reactive oxygen species (ROS) by the NADPH oxidase complex is a critical component of the macrophage bactericidal machinery. We previously characterized brain-specific angiogenesis inhibitor 1 (BAI1), a member of the adhesion family of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs), as a PRR that mediates the selective phagocytic uptake of Gram-negative bacteria by macrophages. We showed that BAI1 promoted phagosomal ROS production through activation of the Rho family guanosine triphosphatase (GTPase) Rac1, thereby stimulating NADPH oxidase activity. Primary BAI1-deficient macrophages exhibited attenuated Rac GTPase activity and reduced ROS production in response to several Gram-negative bacteria, resulting in impaired microbicidal activity. Furthermore, in a peritoneal infection model, BAI1-deficient mice exhibited increased susceptibility to death by bacterial challenge because of impaired bacterial clearance. Together, these findings suggest that BAI1 mediates the clearance of Gram-negative bacteria by stimulating both phagocytosis and NADPH oxidase activation, thereby coupling bacterial detection to the cellular microbicidal machinery.

The axe–txe operon encodes a toxin–antitoxin (TA) pair, Axe–Txe, that was initially identified on the multidrug-resistance plasmid pRUM in Enterococcus faecium. In Escherichia coli, expression of the Txe toxin is known to inhibit cell growth, and co-expression of the antitoxin, Axe, counteracts the toxic effect of Txe. Here, we report the nucleotide sequence of pS177, a 39 kb multidrug-resistant plasmid isolated from vancomycin-resistant Ent. faecium, which harbours the axe–txe operon and the vanA gene cluster. RT-PCR analysis revealed that the axe–txe transcript is produced by strain S177 as well as by other vancomycin-resistant enteroccoci. Moreover, we determine the mechanism by which the Txe protein exerts its toxic activity. Txe inhibits protein synthesis in E. coli without affecting DNA or RNA synthesis, and inhibits protein synthesis in a cell-free system. Using in vivo primer extension analysis, we demonstrate that Txe preferentially cleaves single-stranded mRNA at the first base after an AUG start codon. We conclude that Txe is an endoribonuclease which cleaves mRNA and inhibits protein synthesis.

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Recognition of lipopolysaccharide (LPS) is critical for host defense against Gram-negative bacteria and the regulation of local inflammatory responses at the host-microbial interface. We have previously shown that the adhesion G-protein-coupled receptor (aGPCR) BAI1 acts as a phagocytic pattern recognition receptor (PRR) that selectively recognizes Gram-negative bacteria through a series of five Type I thrombospondin repeats (TSRs) in its extracellular domain. Unlike Toll-like receptor 4 (TLR4), which recognizes the hydrophobic lipid A region of LPS, the BAI1 TSRs bind the core oligosaccharide of canonical LPS structures. In this study, we report that BAI1 requires the phosphorylated inner core L-glycero-d-manno-heptose moiety of LPS for recognition, in the context of intact, live bacteria. Mutant strains of Salmonella enterica serovar Typhimurium or E. coli BW25113 K-12 that fail to phosphorylate the inner core oligosaccharide, specifically heptose I, are not recognized by BAI1. Phosphorylation of inner core oligosaccharides is critical for membrane stability and function and is conserved across most Gram-negative species, thus allowing BAI1 to recognize a broad range of organisms.